System and method for sperm sorting

ABSTRACT

A system and method for sorting sperm is provided. The system includes a housing and a microfluidic system supported by the housing. The system also includes an inlet providing access to the microfluidic system to deliver sperm to the microfluidic system and an outlet providing access to the microfluidic system to harvest sorted sperm from the microfluidic system. The microfluidic system provides a flow path for sperm from the inlet to the outlet and includes at least one channel extending from the inlet to the outlet to allow sperm delivered to the microfluidic system through the inlet to progress along the flow path toward the outlet. The microfluidic system also includes a filter including a first plurality of micropores arranged in the flow path between the inlet and the outlet to cause sperm traveling along the flow path to move against through the filter and gravity to reach the outlet.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on, claims priority to, and incorporatesherein by reference in its entirety for all purposes, U.S. ProvisionalApplication Ser. No. 61/906,740, filed Nov. 20, 2013, and entitled,“SYSTEM AND METHOD FOR SPERM SORTING.”

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

BACKGROUND OF THE INVENTION

The present invention relates generally to systems and methods for spermsorting.

According to estimates, there are more than 70 million infertile couplesworldwide. Approximately 1 in every 4 infertile couples seek clinicaltreatment, where, according to sources, male factor may account forabout 50 percent of the infertility cases. Assisted reproductivetechnology (ARTs), such as in vitro fertilization (IVF),intracytoplasmic sperm injection (ICSI), and intrauterine insemination(IUD, are generally utilized in reproductive clinics to treat infertilecouples. With an increasing rate of male infertility due toenvironmental and physiological conditions, there is an ever growingneed for the use of ARTs in reproductive clinics. Isolation of the mostmotile and morphologically normal sperm is an integral process tocommonly used IVF/ICSI procedures. Selection of healthy sperm fromunprocessed semen (stock sperm) is crucial as it requires selectingsperm that is not only highly motile, but also has a normal morphology,mature nuclei, and lesser reactive oxygen species (ROS) production.Although current IVF/ICSI procedures results in successful pregnancyapproximately 50 percent of the time, the output can be greatlycompromised if the sperm being selected are abnormal.

Currently, the more commonly-known ART techniques use centrifugationbased sperm swim-up, density gradient separation methods, andmicrofluidic based methods with/without the use of chemotaxis to sortsperm. These techniques have potential drawbacks and limitations intheir use for procedures as delicate as IVF/ICSI. It is worth notingthat the centrifugation based sperm sorting techniques, such as swim-up,compromise on sperm quality during the repetitive centrifugation steps.Quality of a sperm sample is degraded during swim-up technique due toROS generation. ROS exposure can greatly harm the DNA of seeminglymotile and healthy sperm. Furthermore, the centrifugation-based spermsorting techniques are labor intensive, and outcome can vary fromtechnician to technician.

Sperm sorting technologies based on microfluidics have an advantagebecause they can precisely handle small volume of sperm samples. On theother hand, microfluidic-based sperm sorting devices have very lowthroughput and can only process small semen volumes, such as 2 μl-50 μl,which limits their application to reproductive clinics, where normalsperm sample can have volume of 1.5 ml.

In a clinical ICSI procedure, an embryologist will have on average 20oocytes that can be handled in four petri dishes, and will need 20sperm. The embryologist would like to choose these 20 sperm in anoligospermic sample among a few hundred sperm. Such scenario wouldrequire real-time monitoring of individual sperm and collection fromoutlet when 20 sperm reach the outlet, which is not attainable usingcurrent clinical or microfluidic technologies. In a second procedure,where an embryologist is handling healthy samples, in vitrofertilization is performed using 0.5 million healthy sperm suspended ina 5-20 μl suspension to be introduced to an oocyte. However, currentsorting systems, such as described above, do not provide the throughputneeded to meet these criteria.

Traditionally, optical microscopes have been used to image sperm forcomputer assisted sperm analysis (CASA) and manual identification ofsperm motility for ARTs. This classical approach has limitations intracking a large number of sperm simultaneously due to its small fieldof view (FOV). In addition, sperm tracking and motility analyses areperformed after sorting. Currently no system exists that can sort andanalyze sperm simultaneously.

It would therefore be desirable to provide a system and method forprocessing, including as sorting, sperm without damaging the sperm orsubjecting the sperm to potentially-damaging conditions. Furthermore, itwould be desirable to provide a system and method that can analyzesperm, but is efficient and able to scale.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding a system and method that integrates micro- and macro-fluidicsto sort sperm in a manner that allows efficient selection of sperm thatare favorably suited to fertilization. In particular, the presentinvention recognizes that sperm suited to fertilization is mostdesirable and can be selected or sorted using a system presents andenvironment that is akin to that presented in the fertilization process.In this regard, a system is provided where macro reservoirs areconnected by micropores to approximate the female genital track. Asystem and method is provide whereby the most motile, morphologicallynormal, mature, and functional sperm pass selectively through themicropores against gravity leaving behind dead or less functional sperm.The present invention is a chemical-free, centrifugation-free, andflow-free technology, where functional sperm are isolated fromunprocessed semen sample with high retrieval rate.

In accordance with one aspect of the invention, a system for sortingsperm is provided that includes a housing and a microfluidic systemsupported by the housing. The system also includes an inlet providingaccess to the microfluidic system to deliver sperm to the microfluidicsystem and an outlet providing access to the microfluidic system toharvest sorted sperm from the microfluidic system. The microfluidicsystem provides a flow path for sperm from the inlet to the outlet andincludes at least one channel extending from the inlet to the outlet toallow sperm delivered to the microfluidic system through the inlet toprogress along the flow path toward the outlet. The microfluidic systemalso includes a filter including a plurality of micropores and arrangedin the flow path between the inlet and the outlet to cause spermtraveling along the flow path to move against the filter and gravity toreach the outlet.

In accordance with another aspect of the invention, a method for sortingsperm is disclosed that includes delivering a sample of sperm to aninlet connected to a microfluidic system and allowing sperm in thesample of sperm to traverse a flow path through the microfluidic systemtoward an outlet providing access to the microfluidic system to harvestsorted sperm from the microfluidic system. The method also includesfiltering the sperm prior to reaching the outlet using a filter having aplurality of micropores and gravity to restrict movement of the spermthrough the filter. The method further includes harvesting sperm passingto the outlet after passing through the filter and overcoming gravity.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plan view of a sperm sorting system in accordance with thepresent invention.

FIG. 1B is a cross-sectional view of the system of FIG. 1A.

FIG. 1C is a schematic view of multichannel system with a collectionchamber to concentrate the sorted sperm.

FIG. 1D is a schematic view of multi-well system with multiple channelsconnecting the inlet and collection or concentration chamber chamber.

FIG. 2A is an exploded, cross-sectional view of a sperm sorting andimaging system in accordance with the present invention.

FIG. 2B is a detailed, perspective view of a microfluidic system ofFIGS. 1A or 2A.

FIG. 2C is a schematic view of a multi-channel microfluidic system inaccordance with the present invention.

FIG. 2D is a cross-sectional view of overall system for sperm sortingand imaging system in accordance with the present invention.

FIG. 3 is a perspective view of a prototype system for sorting sperm inaccordance with the present invention.

FIG. 4 is a series of images of sperm acquired using the presentinvention.

FIG. 5A is graph illustrating motility of human sperm isolated usingdifferent pore diameter filters and retrieved at different time points

FIG. 5B is a graph illustrating retrieval rate of sorted sperm usingdifferent chips.

FIGS. 6A, 6B, and 6C are graphs illustrating curvilinear velocity (VCL),straight line velocity (VSL), and average path velocity (VAP) of stockand sorted sperm using 3, 5, and 8 μm MMSS chips, respectively.

FIG. 7 is a graph showing normal morphology (%) for stock and sortedsperm.

FIG. 8 is a graph showing mature sperm percentage calculated for stockand sorted sperm.

FIG. 9A is a graph showing sperm sorted using 3, 5, and 8 μm filterdevices showed significantly lesser ROS generation compared to swim-upand washing methods.

FIGS. 9B through 9G are reactive oxygen species (ROS) generation graphsfor (B) Semen sample, (C) Washed sperm, (D) Sperm sorted using swim-upmethod (ROS region is highlighted by circle), (E) Sperm sorted using 3μm MMSS chip, (F) Sperm sorted using 5 μm MMSS chip, and (G) Spermsorted using 8 μm MMSS chip.

FIG. 10A is a graph showing sperm sorted using 5 and 8 μm MMSS chipsshowed significantly lesser DNA fragmentation compared to swim-up andunsorted semen sample.

FIGS. 10B through 10F are DNA fragmentation scatter plots for (B) Semensample, (C) Sperm sorted using swim-up method, (D) Sperm sorted using 3μm MMSS chip, (E) Sperm sorted using 5 μm MMSS chip, and (F) Spermsorted using 8 μm MMSS chip.

FIG. 11 is a flow chart setting forth an example of some steps inaccordance with the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention recognizes that the vaginal mucus becomes wateryand forms tiny microchannels that help guide sperm through to the egg.The present invention recognizes exhaustion as a mechanism for sortingsperm and has been experimentally and theoretically demonstrated toleverage exhaustion to sort healthy sperm using coarse-grainedmulti-scale simulation. Specifically, the present invention provides amacro-micro fluidic sperm sorting (MMSS) system to efficiently,reliably, and successfully sort sperm. As will be described, healthymotile sperm is fully collected at the outlets post-sorting. This systemimproves the efficiency of sperm selection process with minimalperturbation, thereby controlling against DNA fragmentation,accumulation of debris, and generation of ROS.

In addition, the present invention can simultaneously sort, monitor, andevaluate sperm. Specifically, the present system enables evaluation ofeach sperm individually, for example, based on velocity response, usinga wide field-of-view (FOV) lensless imaging technology. The systemprovides a microchip-based, wide-FOV, lensless technology utilizingshadow imaging. Additionally, the present invention can be used toharvest morphometrical information, which is a reliable indicator ofmale fertility.

Referring to FIG. 1A, a sperm sorting system 10 is illustrated. Thesystem 10 may be a polydimethylsiloxane-(PDMS) based,polymethylmethacrylate-(PMMA) based, or other microfluidic system. Thesystem 10 includes a housing 12 having an inlet 14 and a collectionchamber 16 having a filter 18 arranged therein. The filter 18 may be apolycarbonate filter or other filter having suitable materialsproperties, such as pore or passage size, as will be described.Referring to FIG. 1B, the inlet 14 and collection chamber 16 areconnected through a passage or flow path 20 extending along amicrofluidic chip 22. As will be described, the microfluidic chip 22 mayinclude a microchip that may be disposable and that handles unprocessedsemen samples (either fresh or frozen, processed or raw), for example of10 μl-3 ml, and sorts sperm rapidly, such as in less than 30 minutes,without the need for complex instrumentation or trained operators.

The flow path 20 extends from the inlet 14 to the collection chamber 16.At the collection chamber 16 a first or bottom chamber 24 is locatedproximate to the microfluidic chip 22 and a second or top chamber 26 islocated distally with respect to the microfluidic chip 22, above thefirst or bottom chamber 24. As will be described, the first chamber 24is designed to collect the semen of a sample, whether fresh or frozen,processed or raw, presented to the inlet 14 and the second chamber 26 isdesigned to filter the motile sperms.

Referring to FIG. 1C, the system described above with respect to FIG. 1Bmay be modified to include an additional collection or “concentration”chamber 25 that is connected to the top chamber by a fluid connection27. That is, in this regard, the sperm may be concentrated in thecollection chamber 25 to facilitate easier harvesting.

In another configuration, as illustrated in FIG. 1D, the collectionchamber 25 may be connected through a plurality of channels 28 eachhaving an inlet 29 opposite the concentration chamber 25. In thisregard, sperm from multiple flow paths 20 of FIG. 1C or from multiplecollection chambers 16 may be delivered to a common concentrationchamber 25. Such variations on the above-described design can be used tofacilitate the use of multiple filters and multiple channels to handleeven larger volumes or for higher throughput applications.

Referring to FIG. 2A, an exploded view of one optional configuration ofthe system 10 that includes an integrated imaging system is illustrated.The imaging system may form a lensless, wide-FOV imaging platform. Inthis view, components of the integrated imaging system, such as a light30, an imaging sensor 31, and a glass protection layer 32 combined withthe above-described system 10. In function, the light 30 illuminatessperm 34 introduced to the microfluidic chip 22 through the inlet 14.The illuminated sperm 34 can be imaged by the imaging sensor 31, whichmay be a charge-coupled device (CCD), complementarymetal-oxide-semiconductor (CMOS), or other imaging device. More,specifically, referring to FIG. 2B, in function, sperm and semen 34 maybe introduced into the outlet 14 using, for example, a pipette 36. Thesperm traverse across a media 38 along the microfluidic chip 22, whichmay include the aforementioned glass 32, as well as a PMMA or othermaterial layer 40, with a double-sided adhesive (DSA) layer 42 arrangedthere between to affix the glass 32 and PMMA layer 40 together.Ultimately, the sperm 34 traverse the microfluidic chip 22 to the outlet16, where a mineral oil 44 may be found. Specifically, a thin layer ofsterile, embryo-tested mineral oil may be placed on top of the media 38in the inlet 14 and outlet 16 to avoid medium evaporation.

As illustrated, different channel lengths may be used or selected foreffective sperm sorting. Furthermore, referring to FIG. 2C, amultichannel design may be utilized where the inlet 14 and collectionchamber 16 are connected by multiple channels 46. As illustrated, a PBScollection buffer 48 may also be included, for example, to use inwashings. Furthermore, the chip substrate housing the channels may bedisposable.

Referring to FIGS. 2A and 2B, if included, lensless imaging can be usedto record the shadow image of each individual sperm 34 onto anoptoelectronic sensor array plane 31. This system 10 targetsdetecting/counting cells or monitoring in real-time the dynamic locationof hundreds of thousands of individual cells on-chip over an ultra-wideFOV, for example, an FOV that is a few centimeters by a few centimeters.This technology provides these features with reduced complexity and easeof miniaturization.

One particular example of the system 10 including the imagingcapabilities is illustrated in FIG. 2D. A standard microscope cannotmonitor a whole microfluidic sorting chip and analyze sperm in realtime. This challenge can be addressed by integrating lensless imagingwith microchannels providing parallel on-chip monitoring and counting ofsperm. The design permits miniaturization of this technology to make itsuitable for an embryology/clinical lab and point-of-care settings.

The system 10, in the example in FIG. 2D, includes the light source 30that is directed through an aperture 50 in the housing 12, such as a 50pm aperture, to focus monochromatic light 52 toward a the microfluidicchip 22, across which the sperm 34 traverse, as described above. Thesystem 10 may be coupled with a computer system 54 connected through adata connection 56, which may be wired or wireless, and a rechargeablebattery or other power source 58 coupled through a power connection 60to provide operational power for the imaging capabilities.

In one configuration, a combination of polymethyl-methacrylate (PMMA) of1.5 mm thickness and double-sided adhesive (DSA) film of 50 μm thicknesscould be used to create microchannels. The DSA film can be cut to createmicrochannels of different lengths ranging from 5 mm to 40 mm using alaser cutter. Inlet and outlet ports extend through the PMMA with adiameter of 0.65 mm and 2 mm, respectively. The DSA film is then placeddirectly onto the PMMA in effect joining the two. A glass slide isplaced onto the other side of the DSA film, such that the height of thechannel is determined by the adhesive layer thickness. The larger outletsize is particularly designed to extract sorted sperm out of the channeleasily accessible by a pipette. The distance between inlet and outletdetermined the channel length. The length of the channel is defined asthe distance between inlet and outlet.

To increase percentage of motile cells at the outlet and for high volumeprocessing, a polycarbonate filter can be integrated into thesemicrochips. This filter-based device can be designed using 3 mm thickPMMA cut to an area of 50 mm by 30 mm and another cut to an area of 30mm by 30 mm. Cylinders of 20 mm diameter can be cut into both PMMAcomponents and align vertically onto one another using 150 μm DSA. A 0.6mm semen injection inlet is also cut into the larger device component ata 10 mm distance. The system can be assembled using a whatman nucleoporefilter located between the two PMMA components.

Referring to FIGS. 1A through 2D, the system 10 can be used inlarge-scale semen processing. To do so, the sperm 34 is introducedthrough the inlet 14 to be place in the microfluidic chip 22. Duringthis movement, the sperm 34 can be imaged using the light 30 and imagingsensor 31. The sperm 34 move toward the outlet 16. Thisoutlet/collection chamber presents two chambers 24, 26. The firstchamber 24 includes a filter presenting micropores and the secondchamber 26 includes another filter including micropores. In this regard,the system 10 presents macro reservoirs 14, 16 connected by microporesto approximate the female genital track. Therein, the sperm 34 movecollectively, influenced by each other, such as would naturally occur,along the medium 38 toward the outlet 16. The most motile,morphologically normal, mature, and functional sperm pass selectivelythrough the micropores against gravity leaving behind dead or lessfunctional sperm in the first chamber 24. That is, the sperm head is ofspherical shape and has size of about 3 μm×4.5 μm. Sperm tails are about45-50 μm long. If a filter having micropores of diameter larger thansperm head is placed in the first and second chambers 24, 26, only spermthat are motile can make their way through the micropores, whereas dead,dying, or damaged sperm cannot pass through the micropores because oftheir long tails. Instead, these dead, dying, and/or damaged spermsuccumb to gravity and remain in the first chamber 24.

Thus, a microchip-based system is provided that is designed such that itdoes not require any centrifugation steps to retrieve healthy, motile,and morphologically normal sperm with minimal ROS generation. The devicedesign makes sperm sorting procedure less labor intensive andinexpensive. The system incorporates utilizes exhaustion inspace-constrained channels as a mechanism for sperm sorting. The systemcan isolate motile and morphologically normal sperm without anycentrifugation step. Thus, a current coarse-grained model of spermmotility is used to model filter-based microfluidic devices in threedimensions, incorporating effects of cooperatively rising fromhydrodynamic interactions between sperm, with channel walls, and withthe filter surfaces and holes. This model allows the design of deviceparameters such as micropore size and incubation times.

The design and operation of the above-described system can be furtherappreciated from the following discussion of one example of a system,configuration for such system, and testing results of such system. Thisis but one example and is non-limiting in nature to the variety ofconfigurations, designs, and operations that may be employed and fallwithin the scope of the present invention.

EXAMPLE Assembly of MMSS Chip

The poly (methyl methacrylate) (PMMA, 3 mm thick; McMaster Carr,Atlanta, Ga.) and double side adhesive (DSA, 120 μm thick, St. Paul,Minn.) were cut using a laser cutter (Versa Laser™, Scottsdale, Ariz.).The design for the chip was generated on Coral Draw4 and implementedonto USLE Engrave software for cutting. Primary components of the MMSSchip included one 3 mm PMMA cut to an area of 50 mm×30 mm (bottomchamber) and another cut to an area of 30 mm×30 mm (top chamber). A 0.6mm injection point was also cut into the bottom PMMA sheet at a 5 mmdistance from the chambers. Cylinders of 20 mm diameter were cut intoboth PMMA components. The bottom PMMA chamber was first attached toglass slide using DSA. Top PMMA chamber was aligned and attached withbottom chamber using DSA. The Nuclepore™ track-etched polycarbonatemembrane filters (Whatman Ltd, 25 mm diameter, 3 μm, 5 μm, 8 μm) weresandwiched between two PMMA chambers during chip assembly. Thus, it wasconsidered that at least 1 μm and less than 10 μm may be a range ofadvantageous pore sizes. A perspective view of the assembled chip isshown in FIG. 3.

Sperm Sorting using MMSS Chip

Thawed, unprocessed semen sample (stock sperm) was injected into theinlet of MMSS chip until it filled the first/bottom chamber. Thefirst/bottom chamber was designed to hold up to 560 μl of the semensample. In another set of experiments, the stock semen sample wasdiluted 4 times with 1 percent bovine serum albumin (BSA) in human tubalfluid (HTF) before injection into MMSS chip. Following injection, thefirst/upper chamber was topped off with 560 μl of 1 percent BSA in HTF.Chips were then stored at 37 degrees C. in incubator for 15, 30, 45, and60 min intervals before fluid from top chamber was collected intoeppendorph tubes for analysis.

Concentration and Motility Analysis

A standard Makler Haemocytometer was used to analyze the sperm samplesfor concentration and motility using optical microscope. Briefly, 1 μlof sperm sample was pipetted onto Makler Haemocytometer and covered withcover-lid provided with Haemocytometer. Sperm were counted by personnelfamiliar with method using a click-counter for at least three times. Thesperm that were moving forward were considered motile.

Viability Analysis

The sperm samples were analyzed for viability using LIVE/DEAD® SpermViability Kit (L-7011, Molecular Probes®). SYBR 14 dye was used to stainlive whereas Propidium Iodide (PI) was used to stain dead sperm. Sampleswere stained according to manufacturer's protocol. Briefly, first SYBR14 dye was added into sperm sample to the final concentration of 100 nM.The sample was incubated for 5 min at 37° C. To stain the dead sperm, PIdye was added to the sample to the final concentration of 10 μM andallowed to incubate for 5 additional min. The sperm samples were smearedon a glass cover slip and imaged using fluorescent microscope Zeiss AxioObserver.Z1. Green and red emission filters were used for SYBR 14 andPI, respectively.

Velocity Measurement

Sperm samples were analyzed using the method described by WHO laboratorymanual for sperm analysis. Briefly, sperm was retrieved from the MMSSchips (3 μm, 5 μm, 8 μm) after 30 min. Slides were prepared by putting 6μl of sperm sample onto a glass slide and covered by using a 18×18 mmcover slip to give the sample a depth of 20.7 μmm. To avoid drying up ofsamples, slides were made periodically, not simultaneously. Each slidewas analyzed under 20× (Carl Zeiss) using light microscopy with liveimages of the sample being projected onto a computer monitor. Using avideo capturing software (Snagit, TechSmith), movement of sperm sampleswere captured at random locations for 5 secs. Videos were converted toimage sequences using VideotoJpeg software at 100 fps. The imagesequence was input into ImageJ (National Institute of Health,http://rsbweb.nih.gov/ij/) for analysis using the CASA plugin to monitorsperm velocity parameters, i.e. straight line velocity (VSL),curvilinear velocity (VCL), and average path velocity (VAP).

Sperm Morphology Assessment

Recovered sperm suspension from 5 μm, and 8 μm MMSS chips were collectedafter 30 mins. Sperm retrieved from 3 μm MMSS chip were not analyzed forsperm morphology, as sperm concentration is too low for morphologyanalysis. A 10 μL sperm suspension was then taken and placed on a cleanand sterile microscope slide and feathered smears were prepared. Smearswere air dried and prepared for fixation. A Spermac staining protocolsimilar to the one provided by FertiPro was followed to stain sperm formorphology assessments. Briefly, dried smears were submerged intoSpermac fixative solution for at least 5 min and then rinsed with DIwater. Stain A was pipetted at one edge of the slides and allowed toflow over the smear. Slides were then placed on a flat surface andallowed to soak with stain for 1 min. The slides were then rinsed withDI water twice. Next, stain B was applied similarly to Stain A andallowed to penetrate sperm for 1 min. This was followed by a singlerinse with DI water. Finally, stain C was pipetted over the smear andallowed to sit for 1 min before rinsing with DI water. At this point, atleast 100 sperm were imaged using oil immersion and 100× objective (N(no. of repeats)=3). The sperm was considered morphologically normal ifit falls within WHO morphology criteria (Head: spherical head; acrosomecovering 40-70% of head area; head length 3.7-4.7 μm; head width 2.5-3.2μm; length-to-width ratio 1.3-1.8; no more than 2 small vacuoles;post-acrosome region should not contain any vacuole. Midpiece: noresidual cytoplasm in midpiece; length of midpiece should beapproximately same as head length; no broken neck. Principal piece: nosharp angles or bends indicative of tail break; thinner than midpiece,length of principal piece should be approximately 10 times the headlength).

Sperm Maturity Assessment

Recovered sperm suspension from 5, and 8 μm MMSS chips were collectedafter 30 mins. Sperm retrieved from 3 μm MMSS chip were not analyzed fornuclear maturity, as sperm concentration is too low for this analysis.Dried smears were fixed with the Spermac fixative solution for 5 min andsubsequently rinsed with DI water. A 5% aniline blue in 4% acetic acidsolution was prepared and was poured over smears. Smears were soaked for5 min in staining solution and then rinsed with DI water. At least 100sperm were assessed using oil immersion 100× objective (N (no. ofrepeats)=3). Sperm heads that stained dark blue were declared immature,while those that remained unstained were considered mature.

ROS Detection

Sperm Washing: 1 ml of semen was removed from a cryopreservation tankand thawed for 15 min in a 37° C. warm bath. Washed semen sample wasprepared by adding 9 ml of HTF+1% BSA media to 1 ml of semen,centrifuging for 500×g for 5 min and removing supernatant while leavingsperm pellet at the bottom of tube. This procedure was repeated threetimes. HTF media was added to sperm pellet and samples were stained withROS studies.

Swim-up Method: 1 ml of semen was removed from a cryopreservation tankand thawed for 15 mins in a 37 desires C. warm bath. The semen wasdiluted with 9 mL of HTF- F+1% BSA. The diluted sperm suspension wasthen centrifuged at 500×g for 5 mins. Following, the supernatant wasremoved and disposed. The remaining pellet was washed again bycentrifuging sample at 500×g for 5 min. The supernatant was removed anddisposed again. Finally, 500 μL of medium was added along the side wallof centrifuge tube while avoiding the disruption of the pellet. Thesample was then placed in the incubator and motile sperm were allowed toswim up out of pellet for 30 min. The motile sperm were collected byleaving pellet behind. MMSS chips were incubated for a 30 mins periodand sperm suspension was recovered for ROS studies.

Staining for ROS detection: ROS generation was examined by using flowcytometry in conjunction with two fluorescent dyes, dyhydroethidium(DHE) and SYTOX green. DHE reacts with the superoxide anion whichproduces two fluorochromes which bind to sperm DNA and produces a redfluorescence. While SYTOX green is indicative of cell viability, itproduces a green fluorescence when the cell is dead. For thisexperiment, four control samples were prepared in which all consisted of200 μL of recovered sperm suspension mixed with 20 μL of hydrogenperoxide. This was followed by an incubation at 37 degrees C. for 30mins. The dyes were added to the samples; no dye for negative control,DHE at 5 μM was added to the second sample, SYTOX green at 50 nM wasadded to the third sample, and the fourth sample contained both DHE andSYTOX at 5 μM and 50 nM respectively. Dyes were incubated for 15 minsand then transferred to the flow cytometer for measurement 15 min priorto test samples. FACSCalibur flow cytometer (Becton Becton Dickinson,San Jose, Calif.) was used during experiments. Argon laser excitation at488 nm was coupled with emission measurements using 530/30 band pass(green) and 585/42 band pass (red) filters for FL1 and FL2,respectively. Non-sperm events were gated out, and at least 10,000 cellswere examined. For test samples, 500 μL sample from thawed semen, theswim up suspension, 3, 5, and 8 μm filter pore size microchips werecollected. DHE and SYTOX at 5 μM and 50 nM respectively were added toeach sample and allowed to incubate for 15 min. Samples were taken tothe flow cytometer for measurement.

DNA Fragmentation

TUNEL assay kit (In Situ Cell Death Detection Kit, Fluorescein by RocheApplied Science) was used to quantify DNA fragmentation for raw semen,swim-up, and retrieved sperm population from microchip devices withfilters of 3, 5 and 8 μm pore size. All these samples were attained aspreviously mentioned in ROS Detection section. Initially, all the spermsuspensions were washed twice by centrifuging at 500×g for 5 min withPBS and 1% BSA. Once washed, the concentrations of sperm cells wereadjusted to 2×10⁶ cells/ml. Sperm suspensions were then fixed with 4%paraformaldehyde in PBS (200 pL for every 100 μL of cell suspension) for30 min at room temperature. Sperm cells were washed twice at 500×g for 6min with PBS and 1% BSA and permeabilized with 0.1% TritonX in 0.1%sodium citrate for 2 min in/on ice. Sperm were washed twice followed by1 hour incubation at 37° C. with 5 μL of enzyme (TdT) solution and 45 μLof label (dUTP-Flourescein) solution. Similarly, a negative and positivecontrol sample was prepared. However, prior to staining, the positivecontrol was incubated with DNase for 40 min at 37° C. During staining,the negative control was only incubated with label solution (withoutenzyme solution). After staining, samples were washed twice with PBS and1% BSA and resuspended in PBS (Muratori et al, 2000). Fluorescenceemission of DNA fragmented cells were assessed with flow cytometer anddetected by the FL-1 detector (521 nm). A total of 5000 events wereacquired. Sperm population was gated out from data to eliminate anysignal from debris. Experiments are repeated 6 times (N=6).

Results and Discussion

To develop a chemical-free and centrifugation-free, high-throughput,vertical sperm sorting device, the MMSS chips were fabricated andassembled as described above. Briefly, it is a two-chamber chipseparated by polycarbonate filters of various diameters, such as, forexample, 3, 5, 8 μm. The sperm sample was injected into the bottomchamber and sorted motile/healthy sperm were collected from the topretrieval chamber. The presence of the filters with, for example,uniform sized pores between two chambers was designed such that the mostmotile and healthy sperm could translocate through the filter pores.Scanning electron microscope (SEM) images of polycarbonate filters usedfor sperm sorting showed uniform pore diameters as shown in FIG. 4. SEMimages of polycarbonate nuclepore track-etched membrane filters ofdifferent micropore diameters, i) 3 μm ii) 5 μm and iii) 8 μm. The scalebar is 10 μm. These images shows the comparative size of various filterpores and sperm.

The sperm head is of spherical shape and has size of about 3 μm×4.5 μm.Sperm tail is about 45-50 μm long. If a filter of diameter larger thansperm head is placed between this two-chamber chip, only sperm which aremotile can make their way through the micropores whereas dead/dyingsperm cannot pass through the micropores because of their long tails.

Sperm Motility and Retrieval Rate

To investigate the motility of the sorted sperm, we analyzed the spermcollected from the top retrieval chamber of all three MMSS chips (3, 5,and 8 μm diameter filter chips). Results showed that the sperm sortedwith MMSS chips showed significantly higher motility as compared tostock sperm sample, such as illustrated in FIG. 5A. Specifically, the 3,5, and 8 μm filter chips showed sperm motility ofgreater-than-or-equal-to 95 percent ±10, greater-than-or-equal-to 90.4percent ±1.8, greater-than-or-equal-to 85.9 percent ±1.5, respectively,which was significantly higher than the stock sperm motility (39.8percent ±1.9). We further investigated the effect of incubation time onsperm motility. Sperm were collected after 15, 30, 45 and 60 mins. Wefound that the motility of the retrieved sperm increased when spermsample was collected after a longer period of time; motility in the caseof 60 mins time point was highest whereas it was lowest for 15 minutestime points, such as illustrated in FIG. 5A. This increased motility isnoticed in all three chips. When HTF+1 percent BSA was pipetted to thetop chamber of the MMSS chip at the start of each experiment, slightturbulence would produce in the sperm sample due to mixing of the twoliquids; stock sperm sample and HTF+1 percent BSA media. This turbulencein sperm sample is the possible reason for the lesser sperm motility atthe start of the experiment (after 15 mins) as compared to latter timepoints (after 30, 45, and 60 mins). In addition, we calculated the spermretrieval rate at various time points, that is, percentage (%) ofhealthy sperm retrieved out of stock sample. Retrieval rate is animportant parameter for any sperm sorting device especially for thesituation where sperm samples have low sperm count (oligospermic andazoospermic specimens). In the MMSS chip, the sperm retrieval rate wasanalyzed over a period of time; 15, 30, 45, and 60 min time points isillustrated in FIG. 5B. Sperm retrieval rate was maximum for samplescollected after 30 mins time points (3.08 percent±0.42, 23.75 percent±3.96, and 28.58 percent ±2.81 for 3, 5, and 8 μm MMSS chips,respectively). We call this 30 minutes time point as a saturation timepoint as sperm retrieval rate was reduced if the sample was incubatedfor more than 30 minutes, such as illustrated in FIG. 5B. We believethat some of the sperm might be travelling back through the filter intobottom chamber after 30 minutes.

Sperm Viability

Motile sperm are considered viable. To substantiate our finding that thesorted sperm are viable, we performed the live/dead staining for sortedsperm for 30 min time point. The viability of sorted sperm wassignificantly higher than stock sperm sample; 41.0 percent ±0.45 (stocksperm), 91.32 percent ±3.43 (3 μm MMSS chip), 89.83 percent ±5.82 (5 μmMMSS chip), 91.59 percent ±4.44 (3 μm MMSS chip).

Effect of Sample Dilution on Sperm Motility and Retrieval

To investigate the effect of sperm sample dilution on motility andretrieval rate, we diluted the stock sperm sample with HTF+1 percent BSAat the ratio of 1:4 before processing using MMSS chips. The motility ofthe sorted sperm was significantly higher than stock sperm sample at all4 time points (15, 30, 45, and 60 mins); 45.8 percent ±1.5 (stocksperm), 95.0 percent ±5.0 (3 μm MMSS chip), 93.7 percent ±4.7 (5 μm MMSSchip), 90.7 percent ±2.5 (8 μm MMSS chip), whereas it was not differentthan if undiluted sperm sample was used, as shown in FIG. 5A. However,the sperm retrieval rate increased if diluted sperm sample is usedinstead of undiluted stock sperm, as shown in FIG. 5B. Maximum retrievalrate was found to be 52.68 percent ±4.97 for 8 μm chip after 30 minutestime point. In diluted sample, sperm has increased mean free path beforehitting another sperm. This phenomena might has helped sperm in reachingand crossing the filter micropore faster. Secondly, the filter has fixednumber of pores (<14 percent porosity). As lesser number of sperm weretrying to cross the filter pores in diluted sample, it was more probablefor each sperm to find an empty pore and translocate through it.

Sperm Velocity Analysis

Various sperm velocity parameters were analyzed, i.e. curvilinearvelocity (VCL), straight line velocity (VSL), and average path velocity(VAP). A representative image of sperm track showing these velocitydefinitions is shown in Supplementary FIG. 3. The original sperm videofrom which FIG. 3 track is generated is given as Supplementary Movie 1.The sorted sperm using MMSS chips showed significantly higher spermvelocities than stock sperm sample, as illustrated in FIG. 6.Specifically, average sperm VCL was increased from 52.7±6.0 μm/sec(stock sperm) to 59.9±3.5 pm/sec, 75.3±3.1 μm/sec, and 75.6±4.5 μm/secfor 3, 5, and 8 μm MMSS chips, respectively, as illustrated in FIG. 6A.Average sperm VSL increased from 44.4±5.6 pm/sec (stock sperm) to52.1±3.5 μm/sec, 63.4±3.5 μm/sec, and 64.1±3.9 μm/sec for 3, 5 and 8 μmchips, respectively, as illustrated in FIG. 6B. Average sperm VAPincreased from 48.4±5.8 μm/sec (stock sperm) to 54.1±3.4 μm/sec,68.0±2.9 μm/sec, and 67.5±4.1 μm/sec for 3, 5, and 8 μm chips,respectively, as illustrated in FIG. 6C. Higher sperm velocitiesindicate that the sorted sperm are healthier than stock sample. When wecompared velocities among the sperm sorted using three different MMSSchips, it was noticed that sperm sorted using 5 and 8 μm MMSS chips gavehigher VCL, VSL, and VAP velocities than 3 μm filter chip. This isprobably due the fact that mostly immature motile sperm having headsizes smaller than 3 μm could pass through the 3 μm micropores. Onlyexception to this was the filter areas where two or more 3 μm pores werejoined together to make up a larger pore.

Sperm Morphological Analysis

For morphological analysis, sperm were stained with Spermac Stain. Spermwere considered morphologically normal based on the strict criteriadefined by WHO. Any sperm sample having >4 percent morphologicallynormal sperm is considered normal. We found that sperm sorted using 5 μmMMSS chips did not improve the sperm quality in term of overallmorphology, though the sorted sperm were motile. Sperm sorted using 8 μmMMSS chips showed significantly improved morphology over stock and spermsorted using 5 μm MMSS chip; 30.0 percent ±7.6 (8 μm MMSS chip), 17.0percent ±3.2 (5 μm MMSS chip), and 17.6 percent ±0.5 (stock sperm).

Sperm Nuclear Maturity Analysis

Sperm were stained with aniline blue and analyzed for nuclear maturity.Aniline blue staining can discriminate the lysine-rich nuclei ofimmature sperm and arginine/cysteine-rich nuclei of mature sperm. Thenuclei of immature sperm were stained with aniline blue and showed acolor contrast between nuclei and acrosome. Representative images ofsperm stained with aniline blue and their assessment criteria is shownin FIG. 7. Sperm sorted using 5 μm filter chip did not show anyimprovement over stock sperm in terms of nuclei maturity. Whereas, spermsorted using 8 μm filter chip showed higher nuclear maturity than stocksperm sample, as shown in FIG. 8.; 40.8 percent ±5.1 (8 μm MMSS chip),25 percent ±4.6 (5 μm MMSS chip), and 26.9 percent ±5.8 (stock sperm).

ROS Generation Analysis

Sorted sperm was analyzed for ROS generation. We have compared the ROSgeneration in the sperm after washing method, conventional swim-upmethod and MMSS chips. We found that sperm sorted by MMSS chips producedsignificantly lesser ROS than swim-up and washing method (FIG. 9). Spermwashing and swim-up method produced ROS in 10.1%±0.3% and 10.6%±1.1% ofthe sperm respectively, whereas sperm sorted using MMSS chips showed ROSproduction in only 0.8%±0.4% (3 μm MMSS chip), 0.7%±0.1% (5 μm MMSSchip) and 1.0%±0.1% (8 μm MMSS chip) of the sperm. Unsorted semen sampleshowed ROS generation in 1.8%±0.6% of the sperm, which clearly indicatedthat the increased generation of ROS in swim-up and washing methods camefrom centrifugation steps.

DNA Fragmentation Analysis

The analysis of sperm DNA fragmentation can differentiate fertile andinfertile men, and sperm samples showing higher level of DNAfragmentation results lower fertilization rates in IVF/ICSI, impairedembryo progression and lower pregnancy rates. Sperm sorted using MMSSchips were analyzed for DNA fragmentation. DNA fragmentation (%) was1.1%±0.3% (8 μm MMSS), 2.1%±0.7% (5 μm MMSS chip), 3.4%±0.8% (3 μm MMSSchip), 3.7%±1.2% (swim-up method), and 31.2%±1.2% (unsorted semen). Thesorted sperm using 5 μm and 8 μm chips showed significantly lower DNAfragmentation (%) than unsorted semen sample and sperm sorted usingswim-up method (FIG. 10).

Discussion

The ideal sperm sorting technique should (i) be rapid andcost-effective, (ii) be less labor intensive, (iii) process larger spermvolumes, (iv) have higher retrieval efficiency to isolate motile spermfrom dead/non-motile sperm, (v) isolate sperm with higher velocity, (vi)isolate morphologically normal and mature sperm, (vii) reduce ROSgeneration and morphological damage by eliminating centrifugation steps,(viii) reduce the percentage of sperm DNA fragmentation. Theseparameters are generally desirable features for any sperm-sorting deviceand the system of the present invention offers a platform providingthese features.

In the particular example provided herein, the total material cost tofabricate one chip is less than a dollar (50 cents for filter, <50 centsfor PMMA and DSA). The MMSS chip rapidly (approximately 30 minutes)isolated motile sperm from non-motile ones with the higher retrievalrate (28.58 percent ±2.81 percent retrieval from stock sperm) thanswim-up technique (<20 percent). The retrieval was further increased to52.68 percent ±4.97 (8 μm filter) by using diluted sample. Althoughsperm dilution gave higher retrieval of healthy sperm, it reduced theactual stock sperm volume that could be processed at a time. The stocksperm may be desirably diluted before processing for (i) low volumeejaculates, and (ii) ejaculated with very low sperm count. MMSS chipdesign is highly scalable and can process large semen volumes by usinglarger filters (for example, 1.5 ml). Processing a large semen sample isneeded to retrieve enough sperm for IVF procedures. Furthermore, highvolume processing is very important for the samples having low spermcount or low sperm motility.

Sperm having higher velocity parameters can increase the ICSIfertilization rates. Sperm sorted by MMSS chip showed significantlyenhanced velocity parameters (VCL, VSL and VAP) compared to stock spermthat clearly demonstrated that sorted sperm were of higher quality.Sperm morphology is another important indicator for a successfulfertilization. Morphologically normal sperm increase the fertilizationrate during IVF procedures. Sorting sperm using 8 μm MMSS chip improvedsperm morphology by 1.7 folds, which is a significant improvement, asillustrated in FIG. 7. It is also interesting to note down theassociation of sperm motility and morphology. We found thatmorphologically normal sperm also showed better velocities, whichdemonstrated that these two functional parameters (sperm velocity andmorphology) are associated.

Sperm nuclear maturity has shown an association with male infertility.Chromatin condensation as described by nuclear maturity is anotherpredictor for IVF outcome. Sperm sorted using 8 μm MMSS chips showedsignificantly improved sperm maturity compared to stock sample, asillustrated in FIG. 8. We also looked into the ROS generation by humansperm. ROS generation is an important investigative tool to assess thesperm quality and its apoptosis status. There are many pathways andreasons leading to sperm ROS generation such as poor differentiationduring spermiogenesis, poor chromatin compactness, exposure to heavymetals, heat or electromagnetic radiations, prolonged in vitro culture,and presence of sperm in the vicinity ROS generating cells. Conventionaltechniques utilizing centrifugation steps to sort healthy sperm isanother reason for ROS generation as these techniques centrifuge spermwith ROS generating cells such as leukocytes. We found that sperm sortedusing all three MMSS chips showed significantly low ROS generationcompared to stock sperm.

DNA fragmentation is another very important indicator for maleinfertility. According to some reports, sperm DNA integrity can beconsidered as an independent marker for fertilization. Sperm sortedusing MMSS chips showed a significant improvement in DNA fragmentationcompared to unsorted semen sample, as shown in FIG. 10. Currently, spermswim-up method is considered standard to sort sperm with lower DNAfragmentation. It is interesting to note here that the sperm sorted with5 and 8 μm MMSS chips showed ever lower DNA fragmentation than swim-upmethod. We believe based on these functional assays that the spermsorted using 8 μm MMSS chip are of better quality compared toconventional methods. The sorting of morphologically normal, mature,motile and functional sperm would potentially improve IVF/ICSI outcomes.

Referring now to FIG. 11, some example steps 100 in a process forsorting sperm are provided. The steps 100, beginning at process block102, include receiving a sample of sperm to an inlet of a microfluidicsystem, such as described above. Thereafter, at process block 104 thesperm of the sample are allowed to traverse a flow path through themicrofluidic system toward an outlet providing access to themicrofluidic system for harvesting of sorted sperm from the microfluidicsystem. At process block 106, the sperm are subjected to a filter priorto reaching the outlet. As described, the filter has a plurality ofmicropores and is oriented restrict movement of the sperm through thefilter using gravity. Thus, at process block 108, sorted sperm isprovided at the outlet. The sorted sperm includes sperm passing to theoutlet after passing through the filter and overcoming gravity.

Thus, the present disclosure provides system and methods for (i)development of a chemical free and flow free system to sort healthysperm, analyze motility, speed and morphology, (ii) isolation of thesorted healthy sperm, and (iii) developing a better understanding ofexhaustion and collective motion of sperm. This platform is aninnovation beyond the existing clinical procedures such as the swim-upand microdrop techniques. It is also novel beyond the reportedmicrofluidic based sperm sorting devices, as it uses a newground-breaking knowledge of exhaustion in space-constrained channelsfor sorting and analyzing sperm. Given that clinical reproductivemedicine has been a challenging field that is labor intensive, such aneasy-to-use microchip can lead to improved selection of healthy spermand decreased dependence on operator skills, facilitating repeatable,and reliable operational steps.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

1. A system for sorting sperm comprising: a housing; a microfluidicsystem supported by the housing; an inlet providing access to themicrofluidic system to deliver sperm to the microfluidic system; anoutlet providing access to the microfluidic system to harvest sortedsperm from the microfluidic system; wherein the microfluidic systemprovides a flow path for sperm from the inlet to the outlet andcomprises: at least one channel extending from the inlet to the outletto allow sperm delivered to the microfluidic system through the inlet toprogress along the flow path toward the outlet; and a filter including aplurality of micropores and arranged in the flow path to cause spermtraveling along the flow path to move against the filter and gravity toreach the outlet.
 2. The system of claim 1 wherein the plurality ofmicropores are sized to permit a head of the sperm to pass therethrough.3. The system of claim 1 wherein the plurality of micropores includemicropores having a size of at least 1 μm.
 4. The system of claim 1wherein the plurality of micropores include micropores having a size ofless than 10 μm.
 5. The system of claim 1 wherein the plurality ofmicropores include micropores that are spherical in shape.
 6. The systemof claim 1 wherein the filter includes a polycarbonate filter.
 7. Thesystem of claim 1 further comprising a collection chamber arranged atthe outlet.
 8. The system of claim 7 wherein the collection chamberincludes two sub-chambers.
 9. The system of claim 8 wherein the filteris arranged within one of the two sub-chambers.
 10. The system of claim8 wherein a first of the two sub-chambers is configured to collect rawsemen associated with the sperm and a second of the two sub-chambers isconfigured to pass motile sperms to the outlet and restrict non-motilesperm.
 11. The system of claim 1 further comprising an imaging systemconfigured to image the sperm within the flow path.
 12. The system ofclaim 1 further comprising an imaging system including at least onelight configured to illuminate the sperm within the flow path and animaging sensor arranged proximate to the flow path to image the sperm.13. The system of claim 11 wherein the imaging sensor includes at leastone of a charge-coupled device (CCD) or complementarymetal-oxide-semiconductor (CMOS) imaging sensor.
 14. The system of claim1 wherein the microfluidic system includes at least one of apolydimethylsiloxane-(PDMS) base or a polymethylmethacrylate-(PMMA)base.
 15. The system of claim 1 wherein the at least one channelincludes a plurality of channels, each of the plurality of channelshaving different channel lengths.
 16. A method for sorting spermcomprising: delivering a sample of sperm to an inlet connected to amicrofluidic system; allowing sperm in the sample of sperm to traverse aflow path through the microfluidic system toward an outlet providingaccess to the microfluidic system to harvest sorted sperm from themicrofluidic system; filtering the sperm prior to reaching the outletusing a filter having micropores and gravity to restrict movement of thesperm through the filter; and harvesting sperm passing to the outletafter passing through the filter and overcoming gravity.
 17. The methodof claim 16 further comprising restricting non-motile sperm fromreaching the outlet using the filter and gravity.
 18. The method ofclaim 16 further comprising restricting non-motile sperm from reachingthe outlet using the filter, another filter, and gravity.
 19. The methodof claim 16 further comprising imaging the sperm within the microfluidicsystem.
 20. The method of claim 16 further comprising imaging the spermwithin the microfluidic system using at least an optical imaging system.